Nanofibrillar cellulose as a phase separation agent

ABSTRACT

Disclosed is the use of a separation agent including fibril cellulose for phase separation, as well as to a method for the separation of at least one liquid phase from at least one phase selected from a second liquid phase, solid phase and semi-solid phase, where the method includes the steps of incorporating a separation agent including fibril cellulose to a mixture where separation of the phases is desired, followed by formation of phases and removing the phases.

FIELD OF THE INVENTION

The invention relates to new applications of fibril cellulose.Particularly the invention relates to the use of fibril cellulose as aseparation agent for interphase separation, as well as for theseparation of solid materials from liquid phase. The invention alsorelates to a phase lock or phase separation agent comprising fibrilcellulose. Further, the invention relates to a method for the separationof liquid phases and solid or semi-solid compounds. The invention alsorelates to a kit or device comprising said separation agent.

BACKGROUND

Many methods and processes in the field of life sciences, molecularbiology, process chemistry and organic chemistry comprise one or moresteps, typically separation or extraction steps where two or morephases, such as organic (non-aqueous) and aqueous phase, or hydrophilicand hydrophobic phase, are required to be separated. It is also oftendesirable to separate solid or semi-solid materials, such as particlesor aggregates from a suspension or mixture to obtain a clear liquid or aliquid containing a desired compound or compounds dissolved therein,which may be isolated from said liquid.

In molecular biology, extraction procedures are typically associated toDNA purification methods. It is often important to obtain pure samplesof plasmids, nuclear DNA and RNA from samples, in assaying for geneswhich express particular proteins, to determine whether or not aparticular sample of cell has been transfected by a foreign gene and soforth. A widely used method for extracting DNA involves differentiatedsolvation of the DNA and the non-DNA material, using phenol andchloroform. The DNA containing sample, typically after severalpreparative steps, is mixed with an organic solvent, usually phenol or amixture of phenol and chloroform. Proteins denaturate and enter theorganic phase (phenol) or precipitate at the interphase between theorganic phase and aqueous phase. The aqueous phase contains the DNA.Mixing of the aqueous phase with alcohol results in the precipitation ofthe DNA, which then can be spooled. Standard protocols for theseparation of the phases require aspiration of the aqueous DNAcontaining solvent. However, both solvents phenol and chloroform areregarded as potentially carcinogenic compounds. Pouring of the materialfrom the second solvent is not acceptable because the barrier betweenphenol and chloroform is not very stable and contamination of phases isinevitable. At the interphase between the layers, it often becomesdifficult to separate the DNA. Frequently poor phase separation andcloudy interphase between the two phases is noticed, which typicallyencumbers the removal of the desired phase.

Some phase lock agents have been proposed for preventing proteininterphase contamination, for easing the phase separation and forming aclearer interphase between the two phase layers.

U.S. Pat. No. 5,106,966 suggests the use of a polyester gel polymer foreasing phase separation and purification of DNA from a biologicalsample.

U.S. Pat. No. 5,175,271 relates to the use of gels based on silica gelpolymers for the same purpose as above.

There is a need for new, rapid and efficient separation agent and phaselock materials to be used particularly for phase separation and forseparation of solid material from liquid, suitable for applications inmolecular biology, process chemistry, life sciences and organicchemistry.

SUMMARY

The invention is directed to the use and to a method of use of aseparation agent comprising fibril cellulose for phase separation. Phaseseparation or interphase separation may comprise any of the followingseparations, where a first liquid phase is separated from at least oneliquid phase immiscible in said first liquid phase: separation ofhydrophilic and hydrophobic phases, separation of organic and inorganicphases, separation of phases having different gravity, and separation ofsolid or semi-solid material from liquid materials, in applications inthe field of molecular biology, process chemistry, life sciences andorganic chemistry.

The invention is further directed to a method for phase separation,where a first liquid phase is separated from at least one phase selectedfrom a second liquid phase immiscible in said first liquid phase, solidphase and semi-solid phase, where the method comprises the steps ofincorporating a separation agent comprising fibril cellulose to amixture where separation of the phases is desired, followed by formationof phases and removing the phases.

In the method a separation agent comprising fibril cellulose is broughtinto contact with a mixture comprising at least one liquid phase and atleast one component insoluble and/or immiscible in said liquid phase,followed by at least one of the steps selected from agitation,centrifugation, and then the separating the phases. Suitably thecomponent is a liquid.

The invention is further directed to a method for the separation ofnucleic acid(s) from a sample containing it, where the sample is broughtinto contact with at least one organic solvent and a separation agentcomprising fibril cellulose, followed by forming and separation ofphases.

Still further, the invention is directed to a kit for use in nucleicacid separation, comprising least one separation agent comprising fibrilcellulose.

Still further, the invention is directed to a device for use in nucleicacid separation, comprising least one separation agent comprising fibrilcellulose.

Further aspects of the invention are directed to a method for theprevention of interphase contamination of molecules in phase separationwhere a separation agent comprising fibril cellulose is incorporated tothe mixture where phase separation is desired.

The characteristic features of the invention are presented in theappended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photo of phase separation in DNA isolation using fibrilcellulose as phase lock agent.

FIG. 2 illustrates graphically B. cereus cell numbers on growth media(TBS) and fibril cellulose (fc) in the beginning (0 h) and afterovernight incubation analyzed by quantitative PCR.

FIG. 3 illustrates graphically quantitative PCR run from C. perfringensdilution series that show strong linear correlation between the dilutionand the PCR response in the presence or absence of 1.5% fibrilcellulose.

FIG. 4 shows viscosity of 0.5% fibril cellulose hydrogels as function ofapplied shear stress in comparison with 0.5% solution of water solublepolymers polyacrylamide (5 000 kDa) and CMC (250 kDa).

FIG. 5 depicts viscosity of 0.5% fibril cellulose hydrogels as functionof measured shear rate in comparison with 0.5% polyacrylamide and CMC.Typical shear rate regions of different physical processes have beenmarked on the figure with arrows

FIG. 6 depicts schematic presentation of a fibril hydrogel containingparticles (cells) flowing in a needle. High shear rate region (lowviscosity) is located at the gel-needle interface and low shear rateregion (very high viscosity) is located in the middle of the needle.

DEFINITIONS

Unless otherwise specified, the terms, which are used in thespecification and claims, have the meanings commonly used in the filedof life sciences, molecular biology, process chemistry and organicchemistry. Specifically, the following terms have the meanings indicatedbelow.

The term “separation agent” refers here to an agent, which effectsand/or enhances the separation of phases, where a first liquid phase isseparated from at least another phase immiscible in said first liquidphase, such as hydrophilic phase and hydrophobic phase, organic phase(non-aqueous) and inorganic phase, phases with different gravities, andalso the separation of liquid phase and one or more components which maybe solid, semi-solid, immiscible or insoluble in said liquid phase, fromeach other. Said separation agent may acts as a barrier between liquidphases and form an interphase between the separated phases. Particularlyin the field of molecular biology a separation agent may also be calledas “phase lock agent” or “phase divider”.

In the broadest sense the expression “phase separation” refers here tothe separation of at least one liquid phase from other phases. Thusphase separation refers to the separation of at least two phases andparticularly to the separation of at least one first liquid phase fromat least another phase selected from a second liquid phase, solid phaseand semi-solid phase. Particularly “phase separation” is understood hereto mean separation of hydrophilic and hydrophobic phases, separation oforganic and inorganic phases, separation of phases having differentgravity, and separation of solid or semi-solid material from liquidmaterials, in applications in the field of molecular biology, processchemistry, life sciences and organic chemistry.

The expression “liquid phase” refers to suspensions, emulsions,solutions and any compounds or mixtures in liquid form.

Interphase is a phase, which is formed between the phases which areseparated. It is formed of the separation agent and water and optionallysolid or semi-solid material contained in the mixture where separationis desired.

The expression “aqueous phase” refers here to a phase, which containswater.

The expression “organic phase” refers here to a phase, which containsone or more non-aqueous organic solvents or organic compounds, suitablyin liquid form.

As used herein, the term “fibril cellulose” is understood to encompassall microfibrillated celluloses (MFC) and nanocelluloses. Further, thereare several other widely used synonyms for fibril cellulose. Forexample: cellulose nanofiber, nanofibrillated cellulose (CNF),nanofibrillar cellulose (NFC), nano-scale fibrillated cellulose,microfibrillar cellulose, or cellulose microfibrils.

In addition, fibril cellulose produced by certain microbes has alsovarious synonymes, for example, bacterial cellulose (BC), microbialcellulose (MC), biocellulose, nata de coco (NDC), or coco de nata (CDN).

DETAILED DESCRIPTION OF THE INVENTION

The aim of the invention was to provide a separation agent for phaseseparation, particularly for applications in the field of molecularbiology, process chemistry, life sciences and organic chemistry, wherethe separation agent aids and enhances the desired separation.

Molecular biology applications refer here for example to the separationof plasmids and nucleic acids (DNA and RNA) from samples, particularlyto extracting DNA from non-DNA material, as well as to generally DNAseparation methods, such as non-organic salting out where the proteincomponents are trapped within the separation agent, typically in theform of a gel, allowing decanting of the aqueous DNA containing phasewithout contamination.

Process chemistry applications encompass for example extraction andseparation methods, particularly the separation of semisolid or solidmaterials from liquid mixtures and suspensions, and also enhancing theseparation of phases. Said applications include also fermentationprocesses.

Applications in organic chemistry particularly comprise for example theseparation of aqueous and organic non-aqueous phases from each other, aswell as the separation hydrophilic and hydrophobic phases from eachother, and the separation of semisolid or solid materials from liquidmixtures and suspensions, and also enhancing the separation of phases.

It was surprisingly found that a separation agent comprising fibrilcellulose provides an excellent means for phase separation, particularlyin processes and methods in the field of molecular biology, processchemistry, life sciences and organic chemistry. The separation agentcomprising fibril cellulose forms a gel in the presence of water. Whensaid separation agent is brought into contact with a mixture comprisingat least one liquid, followed by separation of phases, it forms agel-like barrier or interphase between the phases. The formed phases areorganized into layers according to their specific gravities. This meansthat the mixture is fractionated into separate phases according to theirspecific gravities. When the mixture comprises solid or semi-solidparticles the gel acts as a web which traps the particles within it andsaid particles can then be recovered from the interphase gel.

Fibril cellulose acts efficiently as a separation agent, separationenhancer and as a phase lock agent, it forms readily a sufficientlystable and uniform barrier and provides a clear and sharp interphasebetween the layers.

Fibril cellulose is suitably incorporated in a mixture as an aqueousgel, or as a dry powder, which forms a gel when it is brought intocontact with water or an aqueous polar solvent. A gel may also be formedof fibril cellulose and a polar solvent. Said polar solvent may beselected from lower alkyl alcohols (C1-C8), suitably methanol, ethanol,isopropanol. Suitably water is used.

Fibril cellulose is obtained from any non-animal based cellulose rawmaterial.

The term “cellulose raw material” refers to any cellulose raw materialsource that can be used in production of cellulose pulp, refined pulp,and fibril cellulose.

The cellulose raw material may be based on any plant material thatcontains cellulose or any microbial cellulose.

Plant material may be wood and said wood can be from softwood tree suchas spruce, pine, fir, larch, douglas-fir or hemlock, or from hardwoodtree such as birch, aspen, poplar, alder, eucalyptus or acacia, or froma mixture of softwoods and hardwoods. Non-wood material can be fromagricultural residues, grasses or other plant substances such as straw,leaves, bark, seeds, hulls, flowers, vegetables or fruits from cotton,corn, wheat, oat, rye, barley, rice, flax, hemp, manilla hemp, sisalhemp, jute, ramie, kenaf, bagasse, bamboo or reed.

The cellulose raw material may be also derived from thecellulose-producing micro-organism, such as from bacterial fermentationprocesses. The micro-organisms can be of the genus Acetobacter,Agrobacterium, Rhizobium, Pseudomonas or Alcaligenes, preferably of thegenus Acetobacter and more preferably of the species Acetobacter xylinumor Acetobacter pasteurianus.

The term “cellulose pulp” refers to cellulose fibers, which are isolatedfrom any cellulose raw material using chemical, mechanical,thermo-mechanical, or chemi-thermo-mechanical pulping processes.Cellulose pulp, which can be pulp of plant origin, especially wood(softwood or hardwood pulp, for example bleached birch pulp) and wherethe cellulose molecules are oxidized in one of the above-describedmethods, is easy to disintegrate to fibril cellulose.

The term “fibril cellulose” refers to a collection of isolated cellulosemicrofibrils (nanofibers) or microfibril bundles derived from celluloseraw material. Microfibrils have typically high aspect ratio: the lengthexceeds one micrometer while the number-average diameter is typicallybelow 1000 nm, preferably 1-200 nm. The diameter of microfibril bundlescan also be larger but generally less than 1 μm. The smallestmicrofibrils are similar to so called elementary fibrils, which aretypically 2-12 nm in diameter. The dimensions of the fibrils or fibrilbundles are dependent on raw material and disintegration method.

Fibril cellulose is characterized by very high water retention values, ahigh degree of chemical accessibility and the ability to form stablegels in water and other polar solvents. Fibril cellulose product istypically a dense network of highly fibrillated celluloses. The fibrilcellulose may also contain some hemicelluloses; the amount is dependenton the plant source.

To obtain fibril cellulose mechanical disintegration of cellulose pulp,oxidized cellulose raw material or microbial cellulose is carried outwith suitable equipment such as a refiner, grinder, homogenizer,colloider, friction grinder, ultrasound-sonicator, fluidizer such asmicrofluidizer, macrofluidizer or fluidizer-type homogenizer. Preferablymechanically disintegrated fibril cellulose is used.

Several different grades of fibril celluloses have been developed usingvarious production techniques. The grades have different propertiesdepending on the manufacturing method, degree of fibrillation andchemical composition. The chemical compositions of the grades also vary.Depending on the raw material source, e.g. HW vs. SW pulp, differentpolysaccharide composition exists in the final fibril cellulose product.Typically, non-ionic or native or neutral or non-modified grades havewider fibril diameter while the chemically modified grades are a lotthinner. Size distribution is also narrower for the modified grades.

The “fibril cellulose” refers here to one grade of fibril cellulose or acombination of two or more different grades of fibril cellulose. Forexample modified grades of fibril cellulose may be blended with nativegrade for enhancing binding of certain compounds to the gel or bindingof some specific impurities etc.

The fibril cellulose may be plant derived cellulose or microbial derivedcellulose or any combination thereof. Suitably plant derived nativefibril cellulose is used, preferably as an aqueous gel.

Fibril cellulose is understood to encompass here also any chemically orphysically modified derivates of cellulose, cellulose nanofibers ornanofiber bundles. The chemical modification may be based for example oncarboxymethylation, oxidation, (TEMPO-oxidation), esterification, oretherification reaction of cellulose molecules. Anionic and cationicgrades are examples of chemically modified grades. Modification may alsobe realized by physical adsorption of anionic, cationic, or non-ionicsubstances or any combination of these on cellulose surface. Thedescribed modification can be carried out before, after, or during theproduction of cellulose nanofibers. Modified grades are typicallyprepared from bleached pulps. In the modified grades, the hemicellulosesare also modified together with the cellulose domain. Most probably, themodification is not homogeneous, i.e. some parts are more modified thanothers. Thus, detailed chemical analysis is not possible—the modifiedproducts are always complicated mixtures of different polysaccharidestructures.

Chemically modified grades, such as anionic and cationic gradestypically have their surface charge modified and they may suitably beused as dry powder or an aqueous gel, which is added to a mixture whereseparation is desired. Chemically modified grades may be usedparticularly in the separation of compounds, where a specific surfacecharge enhances the separation. Thus suitable fibril cellulose or acombination of different fibril celluloses may de selected and designedfor this purpose according to the type of the compounds to be separated.

Dry powders of fibril cellulose may conveniently be manufactured byspray drying and/or lyophilization of suspension or dispersionscontaining said fibril cellulose, using any conventional methods knownin the art.

The fibril cellulose gel or hydrogel refers here to an aqueousdispersion of fibril cellulose. The fibril cellulose has excellentgelling ability, which means that it forms a gel already at a lowconsistency in an aqueous medium.

Suitably the cellulose raw material such as cellulose pulp is pretreatedwith acid and base prior to the mechanical disintegration. Thepretreatment is effected by subjecting the cellulose pulp to acidtreatment, preferably with hydrochloric acid for removing any positivelycharged ions having a charge more than +1, followed by treatment with aninorganic base containing positively charged ions having a charge +1,preferably NaOH, where Na+ ions replace the earlier ions. The absence ofany positively charged ions having a charge more than +1 is particularlyadvantageous in life science and molecular biology applications wherecomplex formation of DNA with ions with charges more than +1 can beavoided. The pretreatment provides the final product excellent gellingproperties and transparency. The fibril cellulose obtained frompretreated cellulose raw material is referred to here as ion exchangedfibril cellulose. According to one embodiment of the invention ionexchanged native fibril cellulose is suitably used.

Microbial purity of fibril cellulose is often essential. Therefore,fibril cellulose may be sterilized prior to use, suitably in a gel form.In addition, it is important to minimize the microbial contamination ofthe product before and during the mechanical disintegration, such asfibrillation. Prior to fibrillation/mechanical disintegration, it isadvantageous to aseptically collect the cellulose pulp from the pulpmill immediately after bleaching stage when the pulp is still sterile.

Fibril cellulose hydrogels have typically remarkable high yield stressand high zero-shear viscosity at low concentrations. Thus, i.e. if gasbubbles are generated in the fibril cellulose hydrogels they may staystill for long periods of time. The buoyancy of gas bubbles can be,however, easily increased by lowering gas pressure (e.g. 15 mmHg) abovethe gel, which lowers the solubility of gas in the hydrogels phase and,respectively increases the volumes of initial gas bubbles. The increasedgas bubbles escape easily to upper gas phase.

The separation agent may comprise from 0.05 to 100 wt % of fibrilcellulose. Thus a separation agent comprising 100 wt % of fibrilcellulose particularly refers to the use of fibril cellulose as drypowder.

The separation agent may comprise from 0.05 to 20 wt % of fibrilcellulose in an aqueous dispersion, suitably from 0.1 to 5 wt %,particularly from 0.1 to 3 wt %. The water used may be tap water,deionized water, sterilized water or molecular biology grade high puritywater, depending on the desired application. The aqueous dispersion mayalso comprise at least one polar and water miscible solvent, suitably alower (C1-C8) alcohol, such as methanol, ethanol or isopropanol.

The separation agent may optionally comprise additives, such as coloringagents (for example congored), activated carbon, and the like, suitablyin an amount 0.001-0.5 wt %.

The method for the separation of phases comprises the steps ofincorporating separation agent comprising fibril cellulose to a mixturecomprising the agents, components or compounds, which are separated,followed by formation of phases and removing the phases. The formationof phases comprises one or more steps selected from agitation,centrifuging and settling, depending on field and application.

In the method for the separation of a first liquid phase from at leastone phase selected from a second liquid phase immiscible in said firstliquid phase, solid phase and semi-solid phase, a separation agentcomprising fibril cellulose, suitably in the form of dry powder or anaqueous gel, is incorporated to the mixture where separation of thephases is desired, agitated and allowed to settle whereby the phases andthe interphase are formed and removing the phases

The first liquid phase comprises an aqueous phase, optionally comprisingat least one polar solvent.

The second liquid phase is immiscible in said first liquid phase and itmay comprise organic solvents immiscible in the first liquid phase or amore hydrophobic phase than the first liquid phase.

The removing of phases may comprise one or more steps selected fromdecanting, pipetting, draining, pumping and any other suitable stepsdepending on the field and application where phase separation isdesired.

The separation agent is added to the mixture where separation is desiredin an amount which is sufficient to achieve a uniform layer, which canprovide a barrier between phases. The layer thickness i.e. the thicknessof the fibril cellulose gel is suitably at least 5 μm, suitable forproviding a concentration of 1 mg of fibril cellulose/cm². Naturally thelayer thickness depends on the dimensions of the equipment, particularlythe vessel where the separation is carried out and volumes used in theseparation, from small laboratory diagnostic scale to large industrialscale. The separation agent is aggregated at the interphase and forms abarrier between the phases.

Depending on the application it may be desirable to have stronger gelsproviding a tighter barrier, where the gel may be of heavier grade,having a higher viscosity, or it may be sufficient and desirable to usea gel with lighter grade having a lower viscosity. The viscosity of thegel may be adjusted by varying the concentration of the fibril cellulosein the gel and by selecting a suitable type and grade of fibrilcellulose. The densities of the liquids, such as aqueous and organic,particularly the salt concentration and protein concentration of theaqueous layer have an effect on the separation. Thus the type of theseparation agent may be adjusted and selected with regard toapplication.

Accordingly, the separation agent may be used as such and added to themixture where separation of liquid phases is desired. The rheologicalproperties of the fibril cellulose gel allows it to be pumped, addedwith a syringe etc where needed.

In small scale applications, such as in molecular biology, theseparation agent (phase lock agent), suitably as a gel may beincorporated or packed in ready-to use devices, such as tubes containingthe desired amount of the gel, such as from 0.5 ml to 100 ml.Alternatively, the separation agent (phase lock agent) may beincorporated or packed in syringes, containing the desired amounts ofthe gel, for example from 0.5 ml to 100 ml. The syringe allowsself-contained dispensing into all types of containers, including tubesand micro plates.

These “ready for use” tubes and syringes can be packed, sterilized andstored, and used when desired. These rapid and simple products are idealfor small volume use.

The separation agent may also be incorporated in a kit for applicationin the molecular biology.

For larger scale use, phase lock agent comprising fibril cellulose maybe dispersed in water at the site of use or delivered as a gel insuitable containers.

The method for the separation of nucleic acid (DNA) from a samplecontaining it comprises the steps where the sample in a buffer isbrought into contact with at least one organic solvent and separationagent (phase lock agent) comprising fibril cellulose, followed byforming and separation of phases. Suitably the buffer is an aqueoussaline buffer solution (typically acetate—NaCl) used in the art. Theorganic solvent is a non-aqueous organic solvent, such as phenol andchloroform or a combination thereof. The obtained mixture is added to avessel containing the separation agent or the separation agent is addedto the mixture, followed by forming and separating the phases, i.e. theaqueous phase containing the nucleic acid, the interphase comprising thefibril cellulose gel between the aqueous phase and the organic phase,and the organic phase. The forming and separation is suitably carriedout by centrifuging. Additionally there may be a solid phase comprisingglass beads etc. The nucleic acid containing phase may be removed bypipetting, decanting etc.

For example nucleic acid yield is increased significantly, andinterphase contamination can be prevented. Also a clear improvement inthe purity of the extracted DNA and RNA are due to physical separationfrom the interphase material. It allows easy decantation or pipettingoff of the nucleic acid containing phase. Multiple extractions can becarried out in the same vessel (tube) containing the separation agent aslong as maximum sample volume is not exceeded.

The separation agent present during extractions involving phenol,phenol:chloroform, and chloroform:isoamyl alcohol migrates undercentrifugal force to form a seal between the organic and aqueous phase.The aqueous upper phase containing the nucleic acid material can berecovered even quantitatively by simple decantation to a fresh tube.

During centrifugation, the separation agent forms a strong barrierbetween the aqueous and the organic phases, sealing harmful phenol fumesbeneath this barrier. The aqueous phase can then be recovered simply bydecanting or pipetting. Even minute amounts of plasmid DNA, genomic DNA,DNA from gels, total DNA, Lambda DNA, and phage and M13 DNA can berecovered quickly and safely.

The separation agent is inert material and it does not interfere withstandard nucleic acid restriction or modification enzymes or PCRdetection or PCR based microbial enumeration. In molecular biologydetection, enumeration and quantification of microbes based ontechniques where real-time polymerase chain reaction (PCR) is carriedout, are widely used. In PCR the microbes are broken down to releasetheir DNA and the DNA is thereafter quantified by using specificoligonucleotide primers, thermostable DNA polymerase and appropriatethermal cycler. Typically many polymeric materials inhibit the PCRreactions and make microbial quantification unreliable. The use of theseparation agent comprising fibril cellulose does not interfere withreal time PCR or qPCR. It is essential that the media does not comprisematerials which interfere or inhibit the PCR reactions and makemicrobial quantification unreliable. The separation agent comprisingfibril cellulose does not interact or inhibit PCR detection or PCR basedmicrobial enumeration as can be seen from Examples 2 and 3.

Further, the separation agent is non-toxic material and requires nospecial precautions when used. It provides increased protection and easeof handling when working with organic extraction mixtures.

When using the separation agent comprising fibril cellulose the phaseseparation is easy and cloudy or hazy interphases between the phases canbe avoided. It acts as a barrier between the organic and aqueous phases.A clear interphase is formed between the phase layers. The separationagent eliminates effectively interphase contamination.

The separation agent is heat-stable and can be sterilized and used atelevated temperatures as well.

The long aspect ratio of fibril cellulose provides a firm and steady netstructure of the barrier gel, and further, the gel contains no monomerresidues because in the manufacture of the gel no polymerizationreactions are performed.

The separation agent is suitable for various separation and extractionmethods and processes in the field of molecular biology, processchemistry, life sciences and organic chemistry. It is very useful inanalytics, diagnostics and it is also very valuable in methods utilizedin laboratory robots. The separation agent may suitably be used in theseparation of proteins, such as in the separation of proteins in bloodfor example in diagnostic methods, in the dairy industry, particularlyin the separation of whey proteins from milk, where the separation agentin gel form traps the precipitated molecules and leaves a clear liquidfor further use. In larger scale it can be used for example infermentation processes where rapid and effective separation is needed,for example in brewing industry for the separation of yeast. Theseparation agent can also be used as a “liquid filter” for removing(separating) of beer yeast.

The fibril cellulose may easily be removed from the gel containing theprecipitated particles, for example with enzymes using enzymaticdegradation of cellulose molecules. Proper enzymes are for examplecommercially available cellulases. Alternatively, the gel containingseparation agent and the trapped particles may be diluted with anaqueous or polar liquid; and removing the fibril cellulose bydecantation. The recovered may suitably be used in animal feeds,particularly when no toxic materials are used in the method.

Yields of the separated products are increased as they are not lost inthe interphase and they can be separated more efficiently.

EXAMPLES Example 1. Fibril Cellulose as Separation Agent

Microbial culture (Bacillus cereus) supplemented with 1.5 wt % of nativefibril cellulose (total volume 11 ml, containing 1 ml of growth medium)was mixed with PCR lysis buffer and glass beads. The culture was beatenthoroughly, after which phenol-chloroform was added to mixture andcentrifuged to form phase separation. The result of centrifugation isshown in FIG. 1. The top layer is aqueous layer containing the desiredDNA. The next white narrow phase is the interphase that typicallycontains amphiphilic molecules, such as denatured proteins and variouslipids. The organic phase lies under the interphase and also containsthe pelleted glass beads and other precipitated debris. From FIG. 1 itis evident that fibril cellulose is able to form very sharp and easilyvisible white interphase layer. This layer helps to withdraw, simply bydecanting or pipetting the aqueous phase from the top of the organicphase. Moreover, it also prevents the contamination of aqueous phasewith the interphase material and with the residual organic phasematerial, which easily contaminates the aqueous phase when worked withsmall volumes. The arrow in FIG. 1 points the interphase where fibrilcellulose creates clear and visible layer. The layer locks two phasesand prevents their mixing during the extraction process.

Example 2. Enumeration of Microorganisms from Fibril Cellulose Carrier

Bacillus cereus bacterium was cultivated in two different media, onecontaining 1.5 wt % of native fibril cellulose and one without it, andit was measured if fibril cellulose has effect on sensitivity on PCRassay.

The growth medium used in the experiment was standard trypsin soy broth,and it was prepared according the manufacturer's instructions andautoclaved for sterility. Before autoclaving the growth medium wasdivided into the two parts, one of which 1.5 wt % of fibril cellulosewas added. Small inoculum of B. cereus was grown overnight at 37° C. toreach dense microbial culture, and that was used to inoculate bothgrowth media, about thousand fold dilution was used in the inoculation.Both test media were sampled 0 and 24 hours after the inoculation. 0.1ml sample of culture was used for PCR based microbial enumeration. Theenumeration method was based on the Ruminolyze protocol where themicrobial sample is first diluted to washing buffer, and microbial cellsare pelleted by centrifugation (10 min 18 000×g). The supernatant wasdiscarded and pellet was suspended to enzymatic lysis buffer andthereafter strongly beaten with glass beads in order to bothenzymatically and mechanically lyse microbial cells to release their DNAcontent. The released DNA was purified with phenol-chloroformextraction, then precipitated with ethanol and finally dissolved to theDNA storage buffer. The B. cereus enumeration was performed with two B.cereus selective DNA primers and Sybergreen I chemistry. The results areshown in FIG. 2, where B. cereus cell numbers on growth media (TBS) andfibril cellulose (fc) in the beginning (0 h) and after overnightincubation analyzed by quantitative PCR are shown.

Accordingly Bacillus cereus cell numbers on growth media (TSB) andfibril cellulose (fc) in the beginning (0 h) and after overnightincubation were analyzed by quantitative PCR. The experiment indicatesthat small number of B. cereus cells, about 10⁵ cells/ml, can bedetected in the presence of fibril cellulose that indicates that fibrilcellulose has no effect on the sensitivity of the PCR assay.Furthermore, 24 hour sample gives higher cell numbers than the 0 hoursample. This means that the dynamic range of PCR is not disturbed by thepresence of fibril cellulose and the fibril cellulose does not hinderthe PCR reaction when is it run at its maximum speed, in other wordswhen there are large amounts of template DNA. FIG. 2 also shows thatmore cells are counted in the presence of fibril cellulose at both 0hours and 24 hours sampling points. This suggests that fibril celluloseaids the DNA isolation and provides higher DNA yields for B. cereusenumerations. This suggests that fibril cellulose even improves PCRprotocols.

Similar experiments were also performed with other bacterial species,Clostridium perfringens, Lactobacillus salivarius and Desulfovibriodesulficans. The results were similar to B. cereus findings, and theysupport the contention that the fibril cellulose does not inhibit DNAisolation or PCR procedures.

Example 3. Microbial Quantification in the Presence of Fibril Cellulose

Dilution series (10-, 100 and 1000-fold dilution) of dense Clostridiumperfringens (Cl. per.) culture (about 10⁹ cells/ml) was prepared in thepresence of 1.5 wt % of native fibril cellulose (MFC) and without it.The diluted microbial samples were suspended to lysis buffer and glassbeads and exposed to microbial DNA isolation method (as described inexample 2 above). After the DNA isolation the DNA samples were used forPCR based enumeration by using C. perfringens specific primers andSybergreen I chemistry. The results of PCR quantification are showngraphically in FIG. 3. The data shows strong linear correlation betweenPCR results and calculated dilution without any interference from fibrilcellulose, i.e. between the dilution and the PCR response in thepresence or absence of 1.5 wt % of fibril cellulose.

Example 4. Flow Properties of Fibril Cellulose Hydrogel

The rheological flow properties of fibril cellulose hydrogels showbeneficial several features. The hydrogels have a high viscosity at lowshear (or rest) for optimum suspending capacity of particles but alsoshow shear-thinning behavior at higher shear rates to enable easydispensing and injection. The ability of fibril cellulose to providethese kinds of rheological properties was demonstrated in a test serieswhere the viscosity of fibril cellulose dispersions was measured over abroad shear stress (rate) range in a rotational rheometer (AR-G2, TAInstruments, UK).

Fibril cellulose dispersions (hydrogels) show much higher zero-shearviscosities (the region of constant viscosity at small shear stresses)than other water soluble polymers, as shown in FIG. 4. The zero-shearviscosity of fibril cellulose is greatly increased by smaller fibrildiameter induced by preceding chemical pretreatment of the startingmaterial. The stress at which shear-thinning behavior starts (“yieldstress”) is also considerably high for the fibril cellulose dispersions.The suspending ability of a material is the better the higher the yieldstress. The particles are effectively stabilized against sedimentationby the combined effects of high zero-shear viscosity and high yieldstress and high storage modulus. The gravitational force applied by theparticles is much weaker than the yield stress.

In FIG. 5 the viscosity is presented as a function of the measured shearrate. From this figure it is obvious that the viscosity of the fibrilcellulose dispersions drops at relatively small shear rates and reachesa similar level as that measured for the reference materials at shearrates of about 200 s-1.

The network structure of fibril cellulose breaks down upon shearing(FIG. 5). Upon the application of a certain stress, the viscosity of thesystem drops dramatically and a transition from solid-like toliquid-like behavior occurs.

When two-phase liquids, such as flocculated fibril cellulosedispersions, are sheared (e.g. in a rheometer or in a tube), thedispersed phase tends to move away from the solid boundaries, whichleads to the creation of a lower-viscosity layer of liquid at the wallsof the container (FIG. 6). This phenomenon means that the resistance toflow, i.e. the viscosity is lower at the boundaries than in the bulk ofthe dispersion.

Respectively, pumping, or injection of the fibril cellulose hydrogelwith a syringe and a needle or with pipette is easy even at highconcentrations (1-4%). The phenomenon enables also easy dispensing ofsuspensions with minimum disturbance of the particles (cells in thefigure).

While the invention has been described with respect to specific examplesincluding presently preferred modes of carrying out the invention, thoseskilled in the art will appreciate that there are numerous variationsand permutations of the above described embodiments that fall within thespirit and scope of the invention. It should be understood that theinvention is not limited in its application to the details ofconstruction and arrangements of the components set forth herein.Variations and modifications of the foregoing are within the scope ofthe present invention.

1-28. (canceled)
 29. A method for the separation of a first liquid phasefrom at least one phase selected from a second liquid phase immisciblein said first liquid phase, solid phase and semi-solid phase, saidmethod comprising the steps of incorporating a separation agentcomprising nanofibrillar cellulose to a mixture where separation of thephases is desired, followed by formation of phases and removing thephases.
 30. The method according to claim 29, wherein the nanofibrillarcellulose is plant derived cellulose or microbial derived cellulose orany combination thereof.
 31. The method according to claim 29, whereinthe nanofibrillar cellulose is native or chemically modifiednanofibrillar cellulose, preferably the nanofibrillar cellulose is ionexchanged nanofibrillar cellulose.
 32. The method according to claim 29,wherein the separation agent comprises from 0.1 to 100 wt % ofnanofibrillar cellulose.
 33. The method according to claim 29, whereinthe separation agent comprises from 0.1 to 20 wt % of nanofibrillarcellulose in an aqueous dispersion.
 34. The method according to claim29, wherein the first liquid phase comprises an aqueous phase,optionally comprising at least one polar solvent.
 35. The methodaccording to claim 29, wherein the formation of phases comprise one ormore steps selected from agitation, centrifuging and settling.
 36. Themethod according to claim 29, wherein the removing of phases comprisesone or more steps selected from decanting, pipetting, draining andpumping.
 37. A method for the separation of nucleic acid from a samplecontaining the nucleic acid, said method comprising the steps where thesample is brought into contact with at least one organic solvent and aseparation agent comprising nanofibrillar cellulose, followed by formingand separation of phases.
 38. The method according to claim 37, whereinthe nanofibrillar cellulose is plant derived cellulose or microbialderived cellulose or any combination thereof.
 39. The method accordingto claim 37, wherein the nanofibrillar cellulose is native or chemicallymodified nanofibrillar cellulose, preferably the nanofibrillar celluloseis ion exchanged nanofibrillar cellulose.
 40. The method according toclaim 37, wherein the separation agent comprises from 0.1 to 20 wt % ofnanofibrillar cellulose in an aqueous dispersion.
 41. The methodaccording to claim 37, wherein the sample comprises an aqueous salinebuffer solution and the organic solvent is selected from phenol,chloroform and isoamyl alcohol and combinations thereof.
 42. The methodaccording to claim 37, wherein the forming of phases is carried out bycentrifuging.
 43. A device for phase separation, said device comprisinga tube or syringe and a gel incorporated in said tube or syringe, saidgel comprising an aqueous dispersion of nanofibrillar cellulose.
 44. Thedevice according to claim 43, wherein the nanofibrillar cellulose isplant derived cellulose or microbial derived cellulose or anycombination thereof.
 45. The device according to claim 43, wherein thenanofibrillar cellulose is native or chemically modified nanofibrillarcellulose, preferably the nanofibrillar cellulose is ion exchangednanofibrillar cellulose.
 46. The device according to claim 43, whereinthe tube or syringe comprises from 0.1 to 20 wt % of nanofibrillarcellulose in an aqueous dispersion.
 47. A kit for use in nucleic acidseparation, said kit comprising a separation agent comprisingnanofibrillar cellulose incorporated therein.